KR20110043610A - Biomaterials containing calcium phosphate - Google Patents

Abstract

The present invention relates to biomaterials containing materials containing calcium phosphate, in particular hydroxyapatite or hydroxyapatite, such as biphasic calcium phosphate and calcium phosphate cement, and its suitable for producing implants for bone tissue regeneration or for artificial implants. It is about a use.

Description

Biomaterials containing calcium phosphate {BIOMATERIALS CONTAINING CALCIUM PHOSPHATE}

The main part of the present invention is a novel biomaterial, a method for preparing the same, and an implant, based on calcium phosphate, such as ideal calcium phosphate and calcium phosphate cement, specifically based on hydroxyapatite or based on materials including hydroxyapatite Its use is suitable for the creation of (implants) or for prosthesis for bone tissue regeneration purposes.

Restoration of bone loss, mainly due to trauma and, more rarely due to tumors, is one of the major difficulties encountered by orthopedic surgeons. Small defects ranging from "tight" pseudarthrosis (incomplete merging of visible fractures of component loss) to bone loss of 5-6 cm are often associated with cortical-spongy bone tissue or autologous grafts from cavernous tombs. This is the most common problem (gold standard). Large defects (≧ 6 cm) require even more cumbersome procedures, vascularized bone delivery or Masquelet procedures. Even so, the amount of available autologous bone is limited, bone bonding remains random and these various techniques are a source of so many post-surgical complications at the site of missing implants.

The various biomaterials available in clinical practice can theoretically avoid the disadvantages of autologous transplantation. Unfortunately, none of these are identical to the bone graft results, and they cannot recover large amounts of component loss.

Most of the materials currently studied combine mesenchymal stem cells obtained from bone marrow with biomaterials after several weeks of cell culture in selection and in vitro. This approach is cumbersome and expensive, limiting clinical use.

It is known that coagulated blood promotes bone restoration. L. Okazaki et al ., Clin. Oral Impl. Res., 16 , 2005, 236-243 describe implants based on coagulated blood or desalted bone powder. WO 02/068010 describes bone marrow-based composite materials, which include porous, biocompatible implantable matrices and solidified materials such as bone marrow, blood or plasma coagulum.

These materials resulting from a combination of support and coagulated or non-coagulated blood have been used to date for maxillary facial surgery where the problem of bone bonding is not so important, but little or no has been used for the repair of interosseous bone.

The method for producing these implants requires collecting blood from the donor, most commonly the individual for whom the implant is desired, and then handling the support (dehydrated bone or synthetic polymer, ceramic), in particular mixed with blood. Steps, which are sources of biomaterials. In addition, it is difficult to obtain a homogeneous biomaterial by these methods.

Thus, there remains a need for a method of preparing implantable biomaterials from a support that is synthetic, and therefore easy to produce, that has consistent and homogeneous properties, which provides good quality bone without the use of culturing or sampling steps. It is possible to quickly restore tissue and obtain excellent properties in terms of biocompatibility.

The present invention compensates for the shortcomings of the prior art and can obtain bones of good quality in terms of angiogenesis and hardness to be obtained. In addition, the method of making this biomaterial is simple and easy to implement, does not require a multi-step process for the individual to be treated, and is relatively inexpensive compared to the prior art methods.

Hydroxyapatite is part of the composition of many bone repair materials. Hydroxyapatite may be used alone or in some cases as a mixture with other components in this application, for example in an ideal calcium phosphate or calcium phosphate cement.

Biphasic calcium phosphate, BCP, is used in many medical and dental applications. The ideal calcium phosphate is Nery et al ., J Periodontol. 1992, Sep; 63 (9) : 729-35, first described as a bone repair material. BCP consists of a mixture of hydroxyapatite (HA) Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ and beta tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ) (β-TCP). Its bioactivity and bioabsorption can be controlled through the proportion of β-TCP and hydroxyapatite formed.

US 2005/0226939 describes a method for preparing hydroxyapatite nanoparticles by a method comprising mixing a composition based on calcium ions and phosphate ions and subjecting them to electromagnetic waves. The conditions used in this document do not cause the formation of BCP or hydroxyapatite impregnated in the calcium chloride solution.

BCP-based biomaterials have the advantage of promoting bone production compared to other synthetic biomaterials.

BCP has been a major part of many studies: Lerouxel et al ., Biomaterials, 2006, Sept. 27 (26): 4566-72, 18, 287-294; Malard O. et al ., J. Biomed. Mater. Res., 46 (1), 1999, 103; Mankani MH et al ., Biotechnology and Bioengineering, 72 (1), 2001, 96-107. These various authors made observations related to the size effect of BCP particles. Mankani MH et In al ., the method comprises mixing cells, then reconstituted fibrinogen in CaCl ₂ solution, and thrombin with HA / TCP particles. However, the CaCl ₂ solution is more concentrated than that used in the present invention, and the CaCl ₂ / BCP mole / weight ratio is higher than that used in the biomaterials of the present invention.

Trojani C. et al ., Biomaterials, 27 , 2006, 3256-3264, show good bone transduction for implantation of BCP / Si-hydroxypropylmethylcellulose hydrogel composites with bone marrow cells added with BCP particles calibrated to 40-80 μm. It was shown that this could be obtained. However, this latter method requires the step of taking a bone marrow cell sample and culturing the bone marrow cells.

WO 2006/015275 describes a method for promoting bone regeneration, which comprises the preparation of a composition consisting of calcium phosphate based support material, platelet-rich plasma, PAR receptor activators other than calcium and thrombin. However, the CaCl ₂ concentration in these compositions is so high that if used under the conditions of the present invention it will act as an anticoagulant.

Two components of BCP, HA and β-TCP, represent two major forms of calcium phosphate used in bone and dental surgery. These are remarkably biocompatible and are believed to combine to provide better bioavailability and thus greater efficiency than with HA alone or β-TCP alone. This is because the two components (HA and β-TCP) show synergistic activity in BCP:

As soon as it is implanted in vivo, hydroxyapatite promotes the formation of non-stoichiometric calcium phosphate apatite (known as “biological apatite”) at its surface by epitaxial growth, thanks to its chemical properties can do. This layer of biological apatite very similar to the crystals present in the bone matrix is believed to promote cell adhesion and cell activity.

Much better dissolved than HA, β-TCP maintains supersaturation of calcium and phosphate salts in biological fluids around BCP implants. This may maintain the precipitation of biological apatite on HA. In addition, this phase can absorb much better than HA, thus adjusting the resorption of the implant by changing the HA / β-TCP ratio.

These chemical phenomena have been identified in vitro (JM Bouler, G. Daculsi, Key Engineering Materials 2001; 192-195: 119-122; Yamada S, et al., Biomaterials 1997; 18: 1037-41; S. Yamada et al., J. Biomed. Mater. Res 1997; 37: 346-52) and in in vivo (Daculsi G. et al ., J. Biomed Mater Res 1989; 23: 883-94; G. Daculsi et al ., Int. Rev. Cytol 1997; 129-191). These mechanisms appear to contribute to better clinical efficiency of BCP compared to single-phase HA or TCP materials (Nery EB et al., J Periodontol 1992; 63: 729-35; Ellinger RF et al ., J Periodontics Restorative Dent 1986; 6: 22-33; Passuti N. et al ., Clin Orthop Relat Res 1989; (248): 169-76; Gouin F et al., Rev Chir Orthop Reparatrice Appar Mot 1995; 81: 59-65; Ransford AO et al ., J Bone Joint Surg Br 1998; 80: 13-8; R. Cavagna et al ., J. Long-Term effect of Med. Impl 1999; 9: 403-412).

The present invention is to obtain a biocompatible prosthesis of suitable shape and dimensions by culturing bone cells on the biomaterial of the invention in a form having the shape of the desired prosthesis and culturing the cells under such conditions.

The present invention is based on the discovery that certain calcium phosphates, in particular calcium phosphate apatite, such as hydroxyapatite, inhibit the spontaneous coagulation of whole blood when in contact with whole blood. Specifically, it has been found that HA, and BCPs containing HA, also inhibit spontaneous clotting of whole blood when in contact with whole blood. It has also been found that if hydroxyapatite or BCP is pre-impregnated with physiological saline (aqueous solution of NaCl), as recommended in the instructions using these biomaterials, then the contacted blood does not coagulate.

Experimental conditions that can establish the anticoagulant properties of the support material are described in detail in the experimental section.

A first major part of the invention is a method of making an implantable biomaterial comprising at least one calcium phosphate based support such as hydroxyapatite or a mixture of hydroxyapatite and at least one other material, the method comprising: a support together with at least one coagulant It includes one or more steps of impregnating.

The expression “calcium phosphate based support” refers to hydroxyapatite, fluoroapatite, multisubstituted non-stoichiometric apatite (ms-ns-AP), and one or more calcium selected from other calcium phosphate biomaterials and mixtures of the foregoing: It means the material containing a phosphate apatite type component. Such supports may be composed of hydroxyapatite, BCP, ms-ns-APs and apatite calcium phosphate cements.

The impregnated support is then implanted in the area where the bone defect should be filled. Implantation of the support then causes coagulation of blood in contact with the biomaterial penetrating into the biomaterial in situ. Trials conducted with BCP have observed that BCP mixed with coagulated blood allows for good bone production and to produce bone tissue of very satisfactory quality by a very simple method compared to the prior art case. . If BCP is not impregnated with the coagulant solution, bone formation is not obtained.

According to one variant of the invention, the calcium phosphate based, in particular hydroxyapatite based, or mixture based hydroxyapatite and other materials based supports are implanted in the site where bone defects should be filled and then impregnated in situ with a coagulant solution. do.

Preferably, the support used in the present invention is hydroxyapatite, fluoroapatite, multisubstituted non-stoichiometric apatite (ms-ns-AP) or one of these compounds and one or more other biomaterials such as α and β forms of tricalcium phosphate _{(Ca 3 (PO 4) 2} ), dicalcium phosphate dihydrate (CaHPO ₄ .2H ₂ O), anhydrous dicalcium phosphate (Ca (HPO _4), mono-calcium phosphate monohydrate (Ca (HPO ₄ ) ₂ .H ₂ O), tetracalcium phosphate (Ca (PO ₄ ) ₂ O) and octacalcium phosphate (Ca ₈ H ₂ (PO ₄ ) ₆ ), based on the mixture. Based on apatite or BCP, preferably based on BCP.

Supports that can be used in the present invention, in particular apatite or BCP, can be in any form: corrected or uncorrected granular form or monolithic form.

BCPs that can be used in the present invention consist of high temperature frits. If it is granular, it is ground and corrected according to the selected diameter, for example by screening. Advantageously, the BCP that can be used in the present invention is between 5/95 and 95/5, preferably between 30/70 and 80/20, and advantageously between 40/60 and 60/40 HA (hydroxyapatite ) / β-TCP (β-tricalcium phosphate) by weight / weight ratio of hydroxyapatite and β-tricalcium phosphate.

Advantageously it comprises a porous support, in particular a porous BCP having a pore size in the range of 50 nm to 1000 μm, advantageously 500 nm to 100 μm, and more advantageously 1 μm to 50 μm.

The support used in the present invention, and especially when the BCP is in the form of granules, between 40 and 500 μm, preferably between 40 and 400 μm, even more preferably between 40 and 300 μm, and advantageously between 80 and 200 μm It is advantageous to have a particle diameter in between.

BCP granules or powders are described in Bouler et al ., J Biomed Mater Res, 1996, 32 , 603-609; Bouler et al ., J Biomed Mater Res, 2000, 51 , 680-684; Obadia et al ., J Biomed Mater Res, 2006, 80 (B) , 32-42.

BCP is commercially available from Graftys SARL (Aix-en-Provence).

The hydroxyapatite which can be used in the present invention is preferably in the form of granules. It is obtained commercially from Graftys SARL.

More specifically, the invention relates to a biomaterial comprising a calcium phosphate-based support impregnated with at least one calcium-derived coagulant solution, wherein the support is selected from hydroxyapatite and BCP, and the coagulant is 1 to 50. It is in the form of an aqueous solution having a concentration in the range of mMol / l, and the ratio of the coagulant solution and the HA or BCP is 0.5 to 5 vol / vol. of the coagulant solution relative to the HA or BCP volume.

Preferably, the present invention relates to a biomaterial comprising a calcium phosphate-based support impregnated with at least one calcium-derived coagulant solution, wherein the support is selected from hydroxyapatite and BCP, and the calcium-derived coagulant is HA Or a calcium ratio in the range of 2.5 to 60 μmol per gram of BCP, preferably in the range of 5 to 40 μmol per gram of HA or BCP.

Coagulants include biocompatible calcium salts, such as: CaCl ₂ , Ca (NO ₃ ) ₂ , Ca (EtOAc) ₂ or CaSO _4. Calcium-based coagulant.

Advantageously, the coagulant is calcium-based, which is selected from biocompatible calcium salts, advantageously CaCl ₂ . In order to be able to impregnate the support, in particular HA or BCP as a coagulant, the coagulant is used as an aqueous solution, preferably in a concentration ranging from 1 to 50 mMol / l, preferably 3 to 40 mMol / l, advantageously 5 to 20 It is used as an aqueous solution having a concentration in the range of mMol / l. Especially when the coagulant is a calcium salt, in particular CaCl ₂ , these values are preferred.

The ratio of coagulant solution and HA or BCP used in the process of the present invention is from 0.5 to 5, preferably from 1 to 3, advantageously about 2 volume / volume ratio, relative to the weight of HA or BCP.

According to another variant of the invention, immediately before the implantation of the biomaterial, the biomaterial is prepared immediately by impregnation of the calcium phosphate-based support.

It is also possible to consider preparing the biomaterial of the present invention by applying the following process: the coagulant solution is impregnated with calcium phosphate-based support, which is then dried or lyophilized and then packaged under sterile conditions. And storing it until transplanted.

Advantageously, the impregnation time is 1 minute to 1 hour, preferably 1 minute to 30 minutes, advantageously 5 minutes to 15 minutes.

According to another variant of the invention, the biomaterial of the invention can be prepared by impregnating a calcium phosphate-based support in a coagulant solution, which can then be packaged under sterile conditions and stored until implanted. .

According to one variant of the invention, it is conceivable to combine the (calcium phosphate-based) support biomaterial with a coagulant in powder form. In particular, biomaterials such as BCP or HA in powder or granular form may be mixed with calcium salts in solid powder form. Thus, this biomaterial is stored until used and immediately impregnated with an aqueous solution, for example physiological saline, immediately before implantation in use. Thus, it can be implanted in unimpregnated, dry form.

According to the invention, it is conceivable to add to the support, in particular BCP, one or more optional additives, for example additives such as polymers, ceramic particles, pharmaceutical molecules, bioactive agents, the conditions under which these materials are used There is no adverse effect on their biocompatibility and blood coagulation reaction. If one of these additives has an adverse effect on blood coagulation, it must take into account the amount of coagulant to be used. For example, such additives or active agents may be used by grafting, mixing or impregnating, or coating a support, BCP, and the like. Such additives known to those skilled in the art can modify the rheology or in vivo properties of biomaterials (hardness, absorbency, osteogenicity) or act on the development of infections or inflammatory phenomena (antibiotic, anti-infection). , Anti-inflammatory).

It is also contemplated to introduce one or more therapeutic molecules, such as molecules for preventing or treating a pathological condition selected from cancer and osteoporosis, for example in the biomaterials of the present invention.

It is also contemplated to introduce adipose tissue, or any other tissue or cell preparation, taken from a patient for which the biomaterial is intended, into the biomaterial of the present invention, wherein the adipose tissue or preparation thereof is previously known as blood or plasma or physiological saline. It is suspended in.

In addition, natural or synthetic growth factors can be incorporated into the biomaterials of the present invention. It is also possible to consider the presence of biomarkers or contrast agents that facilitate visualization by resorption of biomaterials and medical imaging of their fate in the organism.

According to the method of the invention, the support, and in particular HA or BCP, is placed in a cavity of a closed sterile container. If the support is granular, for example it can be placed in the internal cavity of the syringe. Appropriate amount of coagulant is introduced into this container, if such a device is used, for example by using a syringe.

If the support, and especially HA or BCP are in monolithic form, it is present in a container of the appropriate shape and size.

In all cases, the volume of the vessel is such that a desired amount of coagulant solution can be introduced.

The closed container containing the support, in particular HA or BCP, and the coagulant, can be stirred to allow homogeneous impregnation of the biomaterial. However, manual impregnation of the support with a coagulant may also be considered.

At the end of this phase, the biomaterial is of the form:

Homogeneous liquid pastes when the support is used in granular form,

When the support is used in monolithic form, the monolith with cavities filled with liquid.

According to one variant of the invention, optionally a mixture with a coagulant in powder form, implanting the support material directly into the space to be filled, and then in the case of a coagulant solution or a mixture already having a coagulant, It may be considered to impregnate it in situ with a suitable aqueous solution. In addition, when in the form of a dry mixture with a coagulant, it may be considered to be implanted without impregnation and left to be impregnated with blood from surrounding tissues.

Another main part of the invention is a biomaterial comprising a calcium phosphate based support such as hydroxyapatite or BCP, impregnated with one or more coagulant solutions as described above.

Depending on the type of device used to make the biomaterial of the present invention and the physical form of the support, HA or BCP, the material can be applied using the means most suitable for the site where bone defects should be filled:

At the end, a syringe is used which includes openings suitable for the particle size and rheology of the biomaterial of the invention, or a tool such as spatula is used. It can also be implanted directly in monolithic form. In the latter case, the monolith will be designed or tailored to correspond to the shape and dimensions of the space in which the shape and dimensions will be filled.

In addition, the main part of the present invention is a method for filling a bone defect, the method comprising the steps listed above, further comprises the step of inserting the biomaterial into the space where the bone defect is recognized. This method may also include tissue-incision and closure steps.

According to the size and arrangement of the bone defects, filling with the biomaterial of the present invention is a temporary bone bonding technique that imparts the necessary mechanical strength to the affected tissue during bone restoration at the site of implantation of the biomaterial of the present invention. Osteosynthesis).

As confirmed by the present inventors, the implantation of the biomaterial of the present invention was able to promote bone tissue formation in a short period of time (weeks), and this bone tissue was very abundantly distributed in blood vessels.

Another main part of the invention consists of a kit for carrying out the method of the invention, the kit comprising a calcium-derived coagulant and calcium phosphate, such as hydroxyapatite, or a mixture of hydroxyapatite and one or more other materials, for example Combinations of microporous BCP, base support. Advantageously, the coagulant is a biocompatible calcium salt such as CaCl ₂ .

The content of coagulant is calculated to compensate for the anticoagulant effect of calcium phosphate, in particular hydroxyapatite and to promote blood coagulation in surrounding tissues.

Such combination may be in the form of a kit comprising:

(a) a sterile device comprising a sterile internal cavity in which a support, for example BCP or HA, is placed,

(b) A sterile reservoir containing a coagulant.

The reservoir (b) is part of the device (a) or a separate entity, such as a tube or bottle from which the coagulant can be taken to deliver the coagulant to the internal cavity of the device (a), or a cavity in which the support is disposed, the coagulant It may be a syringe that can inject.

The internal cavity of the device (a) is of such a size that it is possible to introduce therein the amount of coagulant necessary to produce the biomaterials of the invention and other components of the mixture, for example the active ingredient.

Advantageously, the device (a) also comprises means for applying the biomaterial to the areas where bone defects have been identified.

Such a device may consist of a syringe.

Furthermore, the device disclosed in WO 02/068010 includes a support, in particular a BCP, with a coagulant injected therein and a tube in which a plunger is fitted to release the biomaterial once the biomaterial is formed. Consider using a device such as this.

The biomaterial according to the present invention can be used for the production of bone grafts, whether it is a problem of filling a fracture, a component of trauma or tumor origin, or a defect resulting from a surgical procedure or helping to maintain an implant.

Biomaterials can be introduced by surgical procedures in areas where bone defects must be filled. After the incision, the biomaterial is implanted and the incision is closed.

The biomaterials of the present invention can be combined with bone grafting to allow for temporary merging while waiting for stabilization of the defect area by bone tissue.

Coating the prosthesis with the biomaterial of the present invention may facilitate the implantation of live bone tissue into or around the prosthesis.

The biomaterials of the invention can also be used in vitro or ex vivo as a support for the production of bone tissue:

Specifically, bone cell cultures around this biomaterial allow for the generation of later implantable bone tissue.

Another main aspect of the present invention is the use of in vitro or ex vivo for the production of bone grafts of the biomaterials described above.

The present invention compensates for the shortcomings of the prior art and can obtain bones of good quality in terms of angiogenesis and hardness to be obtained. In addition, the method of making this biomaterial is simple and easy to implement, does not require a multi-step process for the individual to be treated, and is relatively inexpensive compared to the prior art methods. According to the present invention, bone cells can be cultured on the biomaterial of the present invention in a form having a form of an artificial insert to be produced. By culturing the cells under these conditions, a biocompatible prosthesis of appropriate shape and dimensions can be obtained.

1A-1D are procedures for preparing and implanting implants.
FIG. 2 schematically shows the calcium concentration of plasma in contact with calcium phosphate biomaterials.
3-A is a photograph of a product obtained by adding calcium chloride solution to BCP and HA prior to blood addition.
3-B is a photograph of the product obtained by adding an increased calcium chloride solution to BCP.
4 is a scanning electron microscopy analysis of implants consisting of whole blood (A, C) and BCP microparticles (80-200 μm) taken without anticoagulant, or implants (B, D) prepared according to conventional protocols. In implants made without calcium, networks of fibrin and blood clots around the particles are observed (A, C). The white arrow in (C) shows some red blood cells deposited on the particles. Scales A, B: 100 μm; C, D: 10 μm.

Experimental part

I-for coagulation BCP  And HA Effect

1.Principle :

It includes an instant process performed in the operating room. This involves mixing the BCP particles and the coagulant: CaCl ₂ in the polypropylene syringe body. Implanting this biomaterial in areas where bone defects have been identified promotes coagulation around the biomaterial.

2. Materials and Methods

2.1. Ideal Calcium Phosphate Particles:

The biphasic calcium phosphate (BCP) biomaterial consists of 60% hydroxyapatite (HA; Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) and 40% tricalcium phosphate (TCP; Ca ₃ (PO ₄ ) ₂ ) do. BCP particles of size between 40 and 200 microns were provided by Graftys SARL (Merck Provence, France). The particles were sterilized by heating at 180 ° C. for 2 hours.

2.2. Measurement of calcium concentrations in murine and plasma:

Calcium concentrations were measured in plasma of C57BL / 6 mice (Janvier, Le Genest-St-Isle, France). This plasma was prepared from blood collected on heparin by centrifugation at 1800 g for 15 minutes. Heparin is used as an anticoagulant that does not modify plasma calcium levels. Analysis was carried out on a Hitachi automation device (Orleans, France).

2.3. Implant Preparation and Surgical Procedures:

As shown in FIGS. 1A-1D, a syringe 1 comprising a hollow cylindrical body 2 to which the plunger 3 moves is used. At the end of the body 2 which is not blocked by the plunger, the body of the syringe is closed by a filter plug 4. In the body 2 of the syringe, the BCP granules 6 are between the end 5 of the plunger and the filter plug 4 (FIG. 1A). The whole was sterilized before use. The end of the syringe with filter plug 4 is placed in a container 7 filled with an aqueous solution 8 of 1% concentration of CaCl ₂ . The backward movement of the plunger 3 draws this solution 8 into the body 2 of the syringe 1 (FIG. 1B). In order to impregnate the BCP particles into the solution, the whole is held for 10 minutes and then, by the plunger 3, the excess solution 8 of CaCl ₂ is discharged through the filter plug 4 (FIG. 1C). The filter 9 is removed from the filter plug 4 and the pressure on the plunger 3 allows the BCP granules 6 impregnated with the solution 8 onto the working site 10 (FIG. 1D). . Then the graft site is closed again (this step is not shown).

3. Results

3.1. For flocculation Hydroxyapatite  And TCP Effect of:

50 mg of HA powder or TCP powder was placed in a 1 ml ml syringe body. 100 μl of blood was added to each syringe containing either HA or TCP. This mixture was placed on wheels so that the powder was suspended in blood for a coagulation time, ie 10 minutes. In each experiment, one syringe containing 100 μl of whole blood treated as the rest, ie, treated for 10 minutes on a wheel, served as a positive control for coagulation. After 10 minutes, the wheel was stopped, the syringe was recovered, these ends were cut and the blood / powder mixture was pushed out with a syringe plunger to extract. No blood clotting around the powder was observed or observed. Each experiment was repeated three times.

In the presence of 50 mg of HA and 100 μl of whole blood, coagulation was observed to be inhibited. Blood remained liquid.

Control: Positive control for coagulation. Coagulation and serum extrudate were observed.

In the presence of 50 mg TCP + 100 μl of blood, coagulation occurred; This is the result of the formation of an implant, in which the fibrin network keeps the powder homogeneous in suspension.

The same experiment was conducted with calcium chloride added to a syringe containing HA prior to blood introduction: graft formation and coagulation were found.

3.2. For coagulation BCP Effect

It was observed that blood (100 μl) freshly collected in the absence of anticoagulant and immediately mixed with BCP particles (50 mg) did not coagulate. This anticoagulant effect was negated by the addition of CaCl ₂ (20 μl of 1% CaCl ₂ solution), suggesting uptake of plasma calcium by BCP. This hypothesis was confirmed by measuring plasma calcium concentrations before and after contact with BCP. To this end, plasma is prepared from C57BL / 6 mouse blood collected on heparin (an anticoagulant that does not alter plasma calcium concentration). In the presence of BCP, a decrease in plasma calcium concentration of 2.06 ± 0.06 mmol / l (normal value) to 0.59 ± 0.07 mmol / l was observed.

II Effect of Calcium Phosphate-Based Biomaterials on Plasma Calcium Concentration

1.Principle :

50 mg of BCP (60/40) fine particle fractions or 50 mg of HA or β-TCP fractions are contacted with either 50 μl of H ₂ O or 50 μl of 2.5 mM CaCl ₂ H ₂ O solution and at 56 ° C. It was left to dry overnight. These biomaterials were placed in wells of 96-well microplates. An anticoagulant that interferes with calcium levels, 100 μl plasma prepared from C57BL / 6 mouse blood taken from heparin, was added to each well. After 15 minutes of incubation, the plate was centrifuged at 800 g for 2 minutes, the supernatant was collected and the calcium concentration of plasma was measured. Calcium measurements were performed using the QuantiChrom Calcium Assay kit (Centaur, Brussels, Belgium) according to the manufacturer's instructions. To this end, 5 μl of supernatant fractions were contacted with 200 μl of a phenolsulfonphthalein solution, a dye which forms a blue-colored stabilizing complex in the presence of free calcium. After incubation for 3 minutes, a colored intensity measured at 612 nm, which is directly proportional to the calcium concentration in the sample, was obtained. In each plate, a calibration range is prepared using the following calcium concentrations: 0-0.5-1-1.5-2-3-4-5 mM.

2. Results:

It was found that BCP and HA powder in the form of microparticles in contact with plasma significantly and significantly reduced the calcium concentration of the plasma (FIG. 2). The decrease in calcium concentration is similar in BCP and HA, and not observed in β-TCP. Based on the values obtained for plasma alone (1.960 ± 0.044 μm), plasma in the presence of BCP (0.871 ± 0.160 μm) and plasma in the presence of HA (0.840 ± 0.121 μm), calcium absorption is 0.125 μmol of calcium per 50 mg of BCP or HA. Was evaluated.

It was also found that adding 50 μl of 2.5 mM solution (ie 0.125 μmol) to BCP or HA prior to plasma addition could restore normal plasma calcium concentrations (FIG. 2). The same amount of calcium chloride added to β-TCP was added to the first plasma calcium to confirm that there was no absorption by this biomaterial under these conditions.

It was also observed that calcium absorption was the same for the three BCP granules tested, namely 40-80 μm, 80-200 μm, and 200-500 μm fine particles.

In addition, the same compensation results were obtained through the addition of calcium, whether calcium chloride solution was added on the fly in liquid form immediately prior to plasma addition or whether the solution was first dried when in contact with the particles.

III - BCP  And HA of Anticoagulant  Effect of calcium addition

1. Principle:

To demonstrate that a causal relationship exists between the reduction of plasma calcium induced by BCP and HA and the inhibition of coagulation, 50 mg BCP (60/60) after addition of 50 μl 150 mM NaCl or 50 μl 2.5 mM CaCl ₂ H ₂ O 40) A coagulation test was performed on the fraction of the fine particles and the 50 mgHA powder.

2. Results:

After addition of blood without anticoagulant and rotation for 15 minutes, it was found that pre-addition of calcium to BCP and HA can restore the coagulation of blood in contact with these two biomaterials (FIG. 3A).

These results demonstrate that the anticoagulant effects of BCP and HA are actually associated with a decrease in plasma calcium concentration, that these two raw vegetables are induced, and that the addition of calcium can restore coagulation.

The effect of calcium dose-response on coagulation of blood in contact with BCP was analyzed (FIG. 3B). 100 μl of whole blood without anticoagulant supplemented 0.01% (0.68 mM)-0.02% (1.36 mM)-0.05% (3.4 mM)-0.1% (6.8 mM)-0.2% (13.6 mM)-0.5% (34 biomaterial was added to 50 mg of BCP particles in the presence of 50 μl of CaCl ₂ H ₂ O or 150 mM NaCl equivalent volume prepared at a concentration of 1% (68 mM) -10% (680 mM). Prepared. After 15 minutes of incubation on the wheel, the biomaterial was removed from the mold. Here calcium added at low concentrations corresponding to 0.01% and 0.02% could not recover coagulation. The presence of concentrations between 0.05% and 0.5% showed coagulation. Surprisingly, it was found that increasing CaCl ₂ H ₂ O concentration above 1% induces coagulation inhibition once again (FIG. 3B and Table 1).

These experiments showed that there could be an appropriate calcium concentration that could block the anticoagulant effects of BCP 60/40 and that there was an important concentration range that could be respected.

IV . Analysis of Fibrin Networks by Scanning Electron Microscopy

The anticoagulant effect of BCP shown by the absence of cohesive gelling implant formation during the above tests corresponds to the inhibition of the formation of fibrin networks that form a framework of clot at the molecular level. The presence of the fibrin network was analyzed by scanning electron microscopy. For this purpose, 100 μl of blood without anticoagulant was mixed with 50 mg of BCP incubated in the presence of 50 mg of BCP or calcium to prepare an implant and then to dry. After spinning for 15 minutes on the wheel, the mixtures were removed from the mold and immediately immersed in a fixed solution containing 1.6% glutaraldehyde in 0.1 M phosphate buffer, pH 7. The sample was then washed, dehydrated using increased concentrations of alcohol balls, immersed in hexamethyldisilazane (Sigma-Aldrich, L'isle d'Abeau Chesnes, France) for 5 minutes, and dried at room temperature. . It was cupped onto an aluminum support and wrapped with gold / palladium (Polaron, A5100, UK) for 4 minutes and then analyzed using a scanning electron microscope (JEOL 6700F, Japan).

As can be seen in Figure 4, under BCP conditions, no fibrin network between BCP microparticles was observed (Fig. 4A, 4C). The presence of some red blood cells precipitated on the grains proves that the particles mix with the blood. In contrast, in the presence of BCP / calcium, the presence of a clot bearing particles is observed, which is expected to be a mesh of a very large number of red blood cells and fibrin networks (Fig. 4B, 4D).

In a mixture of BCP and blood
Concentration of CaCl ₂ H ₂ O Solution Added
(As% and molar concentrations) Per 50 μl CaCl ₂ H ₂ O
Μmol number of calcium introduced Coagulation test
50 mg BCP + 100 ml blood + 50 ml CaCl ₂ H ₂ O (liquid or dry) 0 - 0.01%
(0.68mM) 0.034 - 0.02% 0.068 + 0.03% 0.102 + 0.04% 0.136 + 0.05% 0.17 + 0.1% 0.34 + 0.2% 0.68 + 0.5% 1.7 + 0.6% 2.04 + 0.7% 2.38 + 0.8% 2.72 + 0.9% 3.06 + One%
(0.68 mM) 3.4 +/- 2% - 10%
(680mM) 34 -

Claims (15)

A biomaterial comprising a calcium phosphate-based support impregnated with a solution of one or more calcium-derived coagulants, wherein the support is selected from hydroxyapatite and BCP (biphasic calcium phosphate): and the coagulant is 1 Wherein the ratio of the coagulant solution to the HA or BCP is from 0.5 to 5 volume / volume ratio of the coagulant solution to the HA or BCP volume. The biomaterial of claim 1, wherein the calcium-derived coagulant is present in a ratio of 2.5 to 60 μmol calcium per HA or BCP gram, and preferably in a range of 5 to 40 μmol calcium per HA or BCP gram. The biomaterial of claim 1, wherein the coagulant is CaCl ₂ . The biomaterial according to any one of claims 1 to 3, wherein the support is in monolithic form. The biomaterial according to any one of claims 1 to 3, wherein the support is in the form of granules. 6. The method of claim 1, further comprising one or more additives selected from: polymers, ceramic particles, pharmaceutical molecules, natural or synthetic growth factors, biomarkers, contrast agents, and tissue or cell preparations. Biomaterials. The biomaterial according to any one of claims 1 to 6, for use as an implant in a method for filling bone defects. Combination of osteosynthesis and the biomaterial according to any one of claims 1 to 7. A kit for carrying out the method of producing a biomaterial according to any one of claims 1 to 7, wherein the kit is a calcium-derived coagulant in the form of an aqueous solution having a concentration in the range of 1 to 50 μm / l, And a combination of a hydroxyapatite (HA) -based or BCP-based support, and the ratio of coagulant solution to HA or BCP is from 0.5 to 5 volume / volume ratio of coagulant solution to HA or BCP volume. The kit of claim 9, wherein said coagulant is CaCl ₂ . The method of claim 9 or 10,
(a) a sterilization device comprising a sterile internal cavity in which the support is disposed,
(b) sterile reservoirs containing coagulant:
Kit comprising a. 12. The kit according to any one of claims 9 to 11, wherein the device (a) comprises means for causing the biomaterial to be applied to the area where bone defects have been identified. The kit as claimed in claim 9, wherein the device (a) consists of a syringe. Use of in vitro or ex vivo of the biomaterial according to any one of claims 1 to 7 as a support for the production of bone tissue. In vitro or ex vivo use of a biomaterial according to any one of claims 1 to 7 for the production of bone grafts.

Images